Herbicide Tolerant Cotton Plants and Methods for Identifying the Same

ABSTRACT

The invention provides specific transgenic cotton plants, plant material and seeds, characterized in that these products harbor a specific transformation event at a specific location in the cotton genome. Tools are also provided which allow rapid and unequivocal identification of the event in biological samples.

FIELD OF THE INVENTION

This invention relates to transgenic cotton plants, plant material andseeds, characterized by harboring a specific transformation event,particularly by the presence of a gene encoding a protein that confersherbicide tolerance, at a specific location in the cotton genome. Thecotton plants of the invention combine the herbicide tolerant phenotypewith an agronomic performance, genetic stability and adaptability todifferent genetic backgrounds equivalent to the non-transformed cottonline in the absence of weed pressure. This invention further providesmethods and kits for identifying the presence of plant materialcomprising specifically transformation event EE-GH3 in biologicalsamples.

BACKGROUND OF THE INVENTION

The phenotypic expression of a transgene in a plant is determined bothby the structure of the gene itself and by its location in the plantgenome. At the same time the presence of the transgene (in a foreignDNA) at different locations in the genome will influence the overallphenotype of the plant in different ways. The agronomically orindustrially successful introduction of a commercially interesting traitin a plant by genetic manipulation can be a lengthy procedure dependenton different factors. The actual transformation and regeneration ofgenetically transformed plants are only the first in a series ofselection steps, which include extensive genetic characterization,breeding, and evaluation in field trials, eventually leading to theselection of an elite event.

Cotton fiber is the single most important textile worldwide. About 80million acres of cotton are harvested annually across the globe. Cottonis the fifth largest crop in the U.S. in terms of acreage production,with over 15 million acres planted in 2000. Primary weed species forcotton are Ipomoea sp. (morning glory), Amaranthus spp. (pigweed),Cyperus spp. (nutsedge), Xanthium spp. (cocklebur) and Sorghum spp.(johnsongrass).

Before the introduction of broad-leaf herbicides that could be used on agrowing cotton field, growers used directed, post-emergence applicationsof nonselective herbicides taking care not to contact the growing cropplants. As this requires a difference in height between the weeds andthe crop, this is not always possible. Especially for small cotton, thispractice is time-consuming and potentially damaging to the crop.

The unequivocal identification of an elite event is becomingincreasingly important in view of discussions on Novel Food/Feed,segregation of GMO and non-GMO products and the identification ofproprietary material. Ideally, such identification method is both quickand simple, without the need for an extensive laboratory set-up.Furthermore, the method should provide results that allow unequivocaldetermination of the elite event without expert interpretation, butwhich hold up under expert scrutiny if necessary. Specific tools for usein the identification of elite event EE-GH3 in biological samples aredescribed herein.

EE-GH3 has been identified as an elite event from a population oftransgenic cotton plants in the development of cotton (Gossypiumhirsutum) resistant to the herbicide N-phosphonomethylglycine and saltsthereof, also known as glyphosate. The transgenic cotton plantscontained a chimeric gene conferring tolerance to glyphosate, comprisinga modified epsps gene from Zea mays encoding glyphosate tolerant5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) (as described inU.S. Pat. No. 6,566,587) under control of a plant-expressible promoter(as described in U.S. Pat. No. 5,491,288 or U.S. Pat. No. 5,792,930).

Cotton plants comprising a glyphosate tolerance gene have been disclosedin the art. However, none of the prior art disclosures fail to teach orsuggest the present invention.

SUMMARY OF THE INVENTION

The present invention relates to a transgenic cotton plant, or seed,cells or tissues thereof, comprising, stably integrated into its genome,an expression cassette which comprises a herbicide tolerance genecomprising the coding sequence of the epsps gene (as described inExample 1.1 herein), which is herbicide tolerant and, in the absence ofweed pressure, has an agronomic performance which is substantiallyequivalent to the non-transgenic isogenic line. Under weed pressure andthe appropriate glyphosate treatment, the plant will have a superioragronomic phenotype compared to the non-transgenic plant.

In the one embodiment of the invention the cotton plant or seed, cellsor tissues thereof comprise elite event EE-GH3.

More specifically, the present invention relates to a transgenic cottonplant, seed, cells or tissues thereof, the genomic DNA of which ischaracterized by the fact that, when analyzed in a PCR identificationprotocol as described herein, using two primers directed to the 5′ or 3′flanking region of EE-GH3 and the foreign DNA, respectively, yields afragment which is specific for EE-GH3. The primers may be directedagainst the 5′ flanking region within SEQ ID NO: 1 and the foreign DNArespectively such as the primers comprising or consisting of thenucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 11 respectively, andyield a DNA fragment of between 300 and 360 bp, preferably of about 334bp.

Reference seed comprising the elite event of the invention has beendeposited at the ATCC under accession number PTA-6878. One embodiment ofthe invention is the seed comprising elite event EE-GH3 deposited asATTC accession number PTA-6878, which will grow into a cotton plantresistant to glyphosate. The seed of ATCC deposit number PTA-xxxx, whichis a seed lot consisting of at least about 95% transgenic kernelshomozygous for the transgene, comprising the elite event of theinvention, which will grow into glyphosate tolerant plants. The seed canbe sown and the growing plants can be treated with glyphosate asdescribed herein to obtain 100% glyphosate tolerant plants, comprisingthe elite event of the invention. The invention further relates tocells, tissues, progeny, and descendants from a plant comprising theelite event of the invention grown from the seed deposited at the ATCChaving accession number PTA-6878. The invention further relates toplants obtainable by propagation of and/or breeding with a cotton plantcomprising the elite event of the invention grown from the seeddeposited at the ATCC having accession number PTA-6878.

The invention further relates to a method for identifying a transgenicplant, or cells or tissues thereof, comprising elite event EE-GH3 whichmethod is based on identifying the presence of characterizing DNAsequences or amino acids encoded by such DNA sequences in the transgenicplant, cells or tissues. According to a preferred embodiment of theinvention, such characterizing DNA sequences are sequences of 15 bp,preferably 20 bp, most preferably 30 bp or more which comprise theinsertion site of the event, i.e. both a part foreign DNA and a part ofthe cotton genome (either the 5′ or 3′ flanking region) contiguoustherewith, allowing specific identification of the elite event.

The present invention further relates to methods for identifying eliteevent EE-GH3 in biological samples, which methods are based on primersor probes which specifically recognize the 5′ and/or 3′ flankingsequence of EE-GH3.

More specifically, the invention relates to a method comprising ofamplifying a sequence of a nucleic acid present in biological samples,using a polymerase chain reaction with at least two primers, one ofwhich recognizes the 5′ or 3′ flanking region of EE-GH3, the other whichrecognizes a sequence within the foreign DNA, preferably to obtain a DNAfragment of between 100 and 500 bp. The primers may recognize a sequencewithin the 5′ flanking region of EE-GH3 (SEQ ID No. 1, from position 1to position 732) or within the 3′ flanking region of EE-GH3 (complementof SEQ ID No 2 from position 431 to position 654) and a sequence withinthe foreign DNA (complement of SEQ ID No 1 from position 733 to 1214 orSEQ ID No 2 from position 1 to position 430), respectively. The primerrecognizing the 5′flanking region may comprise the nucleotide sequenceof SEQ ID No. 3 and the primer recognizing a sequence within the foreignDNA may comprise the nucleotide sequence of SEQ ID No. 11 describedherein.

The present invention more specifically relates to a method foridentifying elite event EE-GH3 in biological samples, which methodcomprises amplifying a sequence of a nucleic acid present in abiological sample, using a polymerase chain reaction with two primershaving the nucleotide sequence of SEQ ID No. 3 and SEQ ID No. 11respectively, to obtain a DNA fragment of about 334 bp.

The present invention further relates to the specific flanking sequencesof EE-GH3 described herein, which can be used to develop specificidentification methods for EE-GH3 in biological samples. Moreparticularly, the invention relates to the 5′ and or 3′ flanking regionsof EE-GH3 which can be used for the development of specific primers andprobes as further described herein. The invention further relates toidentification methods for the presence of EE-GH3 in biological samplesbased on the use of such specific primers or probes. Primers may consistof a nucleotide sequence of 17 to about 200 consecutive nucleotidesselected from the nucleotide sequence of SEQ ID No 1 from nucleotide 1to nucleotide 732 or the complement of the nucleotide sequence of SEQ ID2 from nucleotide 431 to nucleotide 654) combined with primersconsisting of a nucleotide sequence of 17 to about 200 consecutivenucleotides selected from the complement of the nucleotide sequence ofSEQ ID No 1 from nucleotide 733 to nucleotide 1214 or the nucleotidesequence of SEQ ID No 2 from nucleotide 1 to nucleotide 430. Primers mayalso comprise these nucleotide sequences located at their extreme 3′end, and further comprise unrelated sequences or sequences derived fromthe mentioned nucleotide sequences, but comprising mismatches.

The invention further relates to kits for identifying elite event EE-GH3in biological samples, said kits comprising at least one primer or probewhich specifically recognizes the 5′ or 3′ flanking region of EE-GH3.

The kit of the invention may comprise, in addition to a primer whichspecifically recognizes the 5′ or 3′ flanking region of EE-GH3, a secondprimer which specifically recognizes a sequence within the foreign DNAof EE-GH3, for use in a PCR identification protocol. The kits of theinvention may comprise at least two specific primers, one of whichrecognizes a sequence within the 5′ flanking region of EE-GH3, and theother which recognizes a sequence within the foreign DNA. The primerrecognizing the 5′flanking region may comprises the nucleotide sequenceof SEQ ID No. 3 and the primer recognizing the transgene may comprisesthe nucleotide sequence of SEQ ID No. 11 or any other primer asdescribed herein.

The invention further relates to a kit for identifying elite eventEE-GH3 in biological samples, said kit comprising the PCR primers havingthe nucleotide sequence of SEQ ID No. 3 and SEQ ID No. 11 for use in theEE-GH3 PCR identification protocol described herein.

The invention also relates to a kit for identifying elite event EE-GH3in biological samples, which kit comprises a specific probe having asequence which corresponds (or is complementary to) a sequence havingbetween 80% and 100% sequence identity with a specific region of EE-GH3.Preferably the sequence of the probe corresponds to a specific regioncomprising part of the 5′ or 3′ flanking region of EE-GH3. Mostpreferably the specific probe has (or is complementary to) a sequencehaving between 80% and 100% sequence identity to the sequence betweennucleotide 712 to 753 of SEQ ID No 1 or SEQ ID No. 2 from nucleotide 410to 451.

According to another aspect of the invention, DNA sequences aredisclosed comprising the insertion site of the event and sufficientlength of polynucleotides of both the cotton genomic DNA and the foreignDNA (transgene), so as to be useful as primer or probe for the detectionof EE-GH3. Such sequences may comprise at least 9 nucleotides of thecotton genomic DNA and a similar number of nucleotides of the foreignDNA (transgene) of EE-GH3 therewith at each side of the insertion siterespectively. Most preferably, such DNA sequences comprise at least 9nucleotides of the cotton genomic DNA and a similar number ofnucleotides of the foreign DNA contiguous with the insertion site in SEQID NO: 1 or SEQ ID NO: 2.

The methods and kits encompassed by the present invention can be usedfor different purposes such as, but not limited to the following: toidentify the presence or absence of EE-GH3 in plants, plant material orin products such as, but not limited to food or feed products (fresh orprocessed) comprising or derived from plant material; additionally oralternatively, the methods and kits of the present invention can be usedto identify transgenic plant material for purposes of segregationbetween transgenic and non-transgenic material; additionally oralternatively, the methods and kits of the present invention can be usedto determine the quality (i.e. percentage pure material) of plantmaterial comprising EE-GH3.

The invention further relates to the 5′ and/or 3′ flanking regions ofEE-GH3 as well as to the specific primers and probes developed from the5′ and/or 3′ flanking sequences of EE-GH3.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Examples, not intended to limit the invention to specificembodiments described, may be understood in conjunction with theaccompanying Figure, incorporated herein by reference, in which:

FIG. 1: represents schematically the relationship between the citednucleotide sequences and primers. black bar: foreign DNA; light bar: DNAof plant origin; the figures under the bars represent nucleotidepositions; (c) refers to complement of the indicated nucleotidesequence.

FIG. 2: represents results obtained by the PCR Identification protocoldeveloped for EE-GH3. Loading sequence of the gel: Lane1: Molecularweight marker (100 bp ladder); lanes 2 to 4: DNA samples from cottonplants comprising the transgenic event EE-GH3; lanes 5 to 8: DNA samplesfrom transgenic cotton plants not comprising elite event EE-GH3, butcomprising a similar glyphosate tolerance gene; lanes 9 to 11: DNAsamples from transgenic cotton-plants not comprising elite event EE-GH3,and not comprising a glyphosate tolerance gene; lane 12: control DNAsample from wild-type cotton plant; lane 13: no template DNA control;lane 14: molecular weight marker.

FIG. 3: represents results obtained by the zygosity scoring PCR protocoldeveloped for EE-GH3. Loading sequence of the gel: Lane1: Molecularweight marker (100 bp ladder); lanes 2, 4, 6, 7 and 8: DNA samples fromcotton plants comprising the transgenic event EE-GH3 in homozygous form;lanes 3 and 5: DNA samples from cotton plants comprising the transgenicevent EE-GH3 in heterozygous form; lanes 9 and 10: control DNA samplefrom wild-type cotton plant; lane 11: no template DNA control; lane 12:molecular weight marker

DETAILED DESCRIPTION

The incorporation of a recombinant DNA molecule in the plant genometypically results from transformation of a cell or tissue. Theparticular site of incorporation is usually due to “random” integration.

The DNA introduced into the plant genome as a result of transformationof a plant cell or tissue with a recombinant DNA or “transforming DNA”,and originating from such transforming DNA is hereinafter referred to as“foreign DNA” comprising one or more “transgenes”. “Plant DNA” in thecontext of the present invention will refer to DNA originating from theplant which is transformed. Plant DNA will usually be found in the samegenetic locus in the corresponding wild-type plant. The foreign DNA canbe characterized by the location and the configuration at the site ofincorporation of the recombinant DNA molecule in the plant genome. Thesite in the plant genome where a recombinant DNA has been inserted isalso referred to as the “insertion site” or “target site”. Insertion ofthe recombinant DNA into the plant genome can be associated with adeletion of plant DNA, referred to as “target site deletion”. A“flanking region” or “flanking sequence” as used herein refers to asequence of at least 20 bp, preferably at least 50 bp, and up to 5000 bpof DNA different from the introduced DNA, preferably DNA from the plantgenome which is located either immediately upstream of and contiguouswith or immediately downstream of and contiguous with the foreign DNA.Transformation procedures leading to random integration of the foreignDNA will result in transformants with different flanking regions, whichare characteristic and unique for each transformant. When therecombinant DNA is introduced into a plant through traditional crossing,its insertion site in the plant genome, or its flanking regions willgenerally not be changed. An “insertion region” as used herein refers tothe region corresponding to the region of at least 40 bp, preferably atleast 100 bp, and up to 10000 bp, encompassed by the sequence whichcomprises the upstream and/or the downstream flanking region of aforeign DNA in the plant genome. Taking into consideration minordifferences due to mutations within a species, an insertion region willretain, upon crossing into a plant of the same species, at least 85%,preferably 90%, more preferably 95%, and most preferably 100% sequenceidentity with the sequence comprising the upstream and downstreamflanking regions of the foreign DNA in the plant originally obtainedfrom transformation.

An event is defined as a (artificial) genetic locus that, as a result ofgenetic engineering, carries a transgene comprising at least one copy ofa gene of interest. The typical allelic states of an event are thepresence or absence of the foreign DNA. An event is characterizedphenotypically by the expression of the transgene. At the genetic level,an event is part of the genetic make-up of a plant. At the molecularlevel, an event can be characterized by the restriction map (e.g. asdetermined by Southern blotting), by the upstream and/or downstreamflanking sequences of the transgene, the location of molecular markersand/or the molecular configuration of the transgene. Usuallytransformation of a plant with a transforming DNA comprising at leastone gene of interest leads to a population of transformants comprising amultitude of separate events, each of which is unique.

An elite event, as used herein, is an event which is selected from agroup of events, obtained by transformation with the same transformingDNA or by back-crossing with plants obtained by such transformation,based on the expression and stability of the transgene(s) and itscompatibility with optimal agronomic characteristics of the plantcomprising it. Thus the criteria for elite event selection are one ormore, preferably two or more, advantageously all of the following:

a) That the presence of the foreign DNA does not compromise otherdesired characteristics of the plant, such as those relating toagronomic performance or commercial value;b) That the event is characterized by a well defined molecularconfiguration which is stably inherited and for which appropriate toolsfor identity control can be developed;c) That the gene(s) of interest show(s) a correct, appropriate andstable spatial and temporal phenotypic expression, both in heterozygous(or hemizygous) and homozygous condition of the event, at a commerciallyacceptable level in a range of environmental conditions in which theplants carrying the event are likely to be exposed in normal agronomicuse.

It is preferred that the foreign DNA is associated with a position inthe plant genome that allows easy introgression into desired commercialgenetic backgrounds.

The status of an event as an elite event is confirmed by introgressionof the elite event in different relevant genetic backgrounds andobserving compliance with one, two or all of the criteria e.g. a), b)and c) above.

An “elite event” thus refers to a genetic locus comprising a foreignDNA, which answers to the above-described criteria. A plant, plantmaterial or progeny such as seeds can comprise one or more elite eventsin its genome.

The tools developed to identify an elite event or the plant, plantmaterial comprising an elite event, or products which comprise plantmaterial comprising the elite event are based on the specific genomiccharacteristics of the elite event, such as, a specific restriction mapof the genomic region comprising the foreign DNA, molecular markers orthe sequence of the flanking region(s) of the foreign DNA.

Once one or both of the flanking regions of the foreign DNA have beensequenced, primers and probes can be developed which specificallyrecognize this (these) sequence(s) in the nucleic acid (DNA or RNA) of asample by way of a molecular biological technique. For instance a PCRmethod can be developed to identify the elite event in biologicalsamples (such as samples of plants, plant material or productscomprising plant material). Such a PCR is based on at least two specific“primers” one recognizing a sequence within the 5′ or 3′ flanking regionof the elite event and the other recognizing a sequence within theforeign DNA. The primers preferably have a sequence of between 15 and 35nucleotides which under optimized PCR conditions “specificallyrecognize” a sequence within the 5′ or 3′ flanking region of the eliteevent and the foreign DNA of the elite event respectively, so that aspecific fragment (“integration fragment” or discriminating amplicon) isamplified from a nucleic acid sample comprising the elite event. Thismeans that only the targeted integration fragment, and no other sequencein the plant genome or foreign DNA, is amplified under optimized PCRconditions.

PCR primers suitable for the invention may be the following:

-   -   oligonucleotides ranging in length from 17 nt to about 200 nt,        comprising a nucleotide sequence of at least 17 consecutive        nucleotides, preferably 20 consecutive nucleotides selected from        the 5′ flanking sequence (SEQ ID No 1 from nucleotide 1 to        nucleotide 732) at their 3′ end (primers recognizing 5′ flanking        sequences); or    -   oligonucleotides ranging in length from 17 nt to about 200 nt,        comprising a nucleotide sequence of at least 17 consecutive        nucleotides, preferably 20 consecutive nucleotides, selected        from the 3′ flanking sequence (complement of SEQ ID No 2 from        nucleotide 431 to nucleotide 654) at their 3′ end (primers        recognizing 3′ flanking sequences); or    -   oligonucleotides ranging in length from 17 nt to about 200 nt,        comprising a nucleotide sequence of at least 17 consecutive        nucleotides, preferably 20 nucleotides selected from the        inserted DNA sequences (complement of SEQ ID No 1 from        nucleotide 733 to nucleotide 1214) at their 3′ end (primers        recognizing foreign DNA) or    -   oligonucleotides ranging in length from 17 nt to about 200 nt,        comprising a nucleotide sequence of at least 17 consecutive        nucleotides, preferably 20 nucleotides selected from the        inserted DNA sequences (SEQ ID No 2 from nucleotide 1 to        nucleotide 430)

The primers may of course be longer than the mentioned 17 consecutivenucleotides, and may e.g. be 20, 21, 30, 35, 50, 75, 100, 150, 200 ntlong or even longer. The primers may entirely consist of nucleotidesequence selected from the mentioned nucleotide sequences of flankingsequences and foreign DNA sequences. However, the nucleotide sequence ofthe primers at their 5′ end (i.e. outside of the 3′-located 17consecutive nucleotides) is less critical. Thus, the 5′ sequence of theprimers may consist of a nucleotide sequence selected from the flankingsequences or foreign DNA, as appropriate, but may contain several (e.g.1, 2, 5, 10 mismatches). The 5′ sequence of the primers may evenentirely consist of a nucleotide sequence unrelated to the flankingsequences or foreign DNA, such as e.g. a nucleotide sequencerepresenting restriction enzyme recognition sites. Such unrelatedsequences or flanking DNA sequences with mismatches should preferably benot longer than 100, more preferably not longer than 50 or even 25nucleotides.

Moreover, suitable primers may comprise or consist of a nucleotidesequence at their 3′ end spanning the joining region between the plantDNA derived sequences and the foreign DNA sequences (located atnucleotides 732-733 in SEQ ID No 1 and nucleotides 430-431 in SEQ ID No2) provided the mentioned 3′-located 17 consecutive nucleotides are notderived exclusively from either the foreign DNA or plant-derivedsequences in SEQ ID No 1 or 2.

It will also be immediately clear to the skilled artisan that properlyselected PCR primer pairs should also not comprise sequencescomplementary to each other. For the purpose of the invention, the“complement of a nucleotide sequence represented in SEQ ID No: X” is thenucleotide sequence which can be derived from the represented nucleotidesequence by replacing the nucleotides through their complementarynucleotide according to Chargaff's rules (A

T; G

C) and reading the sequence in the 5′ to 3′ direction, i.e in oppositedirection of the represented nucleotide sequence.

Examples of suitable primers are the oligonucleotide sequences of SEQ IDNo 3 (5′ flanking sequence recognizing primers), SEQ ID No 4, SEQ ID No5 SEQ ID No 6 (foreign DNA recognizing primers for use with the 5′flanking sequence recognizing primers), SEQ ID No 7, SEQ ID No 8, SEQ IDNo 9 (foreign DNA recognizing primers for use with the 3′ flankingsequence recognizing primers), or SEQ ID No 10 (3′ flanking sequencerecognizing primers).

Other examples of suitable oligonucleotide primers comprise at their 3′end the following sequences or consist of such sequences:

a. 5′ flanking sequence recognizing primers:

-   -   the nucleotide sequence of SEQ ID No 1 from nucleotide 258 to        nucleotide 277    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 310 to        nucleotide 329    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 311 to        nucleotide 330    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 507 to        nucleotide 526    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 616 to        nucleotide 635    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 256 to        nucleotide 275    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 259 to        nucleotide 277    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 260 to        nucleotide 279    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 309 to        nucleotide 329    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 310 to        nucleotide 330    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 311 to        nucleotide 329    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 312 to        nucleotide 330    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 436 to        nucleotide 455    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 34 to        nucleotide 53    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 130 to        nucleotide 148    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 139 to        nucleotide 158    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 256 to        nucleotide 277    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 259 to        nucleotide 279    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 260 to        nucleotide 277    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 261 to        nucleotide 279    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 266 to        nucleotide 285    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 308 to        nucleotide 329    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 309 to        nucleotide 330    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 312 to        nucleotide 329    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 313 to        nucleotide 330    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 435 to        nucleotide 455    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 501 to        nucleotide 518    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 509 to        nucleotide 526    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 513 to        nucleotide 532    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 528 to        nucleotide 547    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 529 to        nucleotide 547    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 602 to        nucleotide 621    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 21 to        nucleotide 40    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 35 to        nucleotide 54    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 129 to        nucleotide 149    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 134 to        nucleotide 153    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 138 to        nucleotide 158    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 140 to        nucleotide 158    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 150 to        nucleotide 169    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 186 to        nucleotide 205    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 254 to        nucleotide 275    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 258 to        nucleotide 279    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 262 to        nucleotide 279    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 434 to        nucleotide 455    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 512 to        nucleotide 532    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 515 to        nucleotide 532    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 530 to        nucleotide 547    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 22 to        nucleotide 40    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 32 to        nucleotide 53    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 34 to        nucleotide 54    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 82 to        nucleotide 99    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 128 to        nucleotide 149    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 135 to        nucleotide 153    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 137 to        nucleotide 158    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 141 to        nucleotide 158    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 141 to        nucleotide 161    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 168 to        nucleotide 187    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 185 to        nucleotide 205    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 187 to        nucleotide 205    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 264 to        nucleotide 285    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 511 to        nucleotide 532    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 522 to        nucleotide 541    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 527 to        nucleotide 547    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 19 to        nucleotide 40    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 22 to        nucleotide 43    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 23 to        nucleotide 40    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 33 to        nucleotide 54    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 132 to        nucleotide 153    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 136 to        nucleotide 153    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 140 to        nucleotide 161    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 150 to        nucleotide 171    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 169 to        nucleotide 188    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 169 to        nucleotide 187    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 171 to        nucleotide 189    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 173 to        nucleotide 193    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 184 to        nucleotide 205    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 188 to        nucleotide 205    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 521 to        nucleotide 541    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 523 to        nucleotide 541    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 602 to        nucleotide 623    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 97 to        nucleotide 118    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 168 to        nucleotide 188    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 170 to        nucleotide 187    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 170 to        nucleotide 188    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 172 to        nucleotide 189    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 172 to        nucleotide 193    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 520 to        nucleotide 541    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 91 to        nucleotide 110    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 167 to        nucleotide 188    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 171 to        nucleotide 188    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 176 to        nucleotide 197    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 90 to        nucleotide 110    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 92 to        nucleotide 110    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 300 to        nucleotide 319    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 89 to        nucleotide 110    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 93 to        nucleotide 110    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 298 to        nucleotide 319        b. foreign DNA sequence recognizing primers for use with 5′        flanking sequence recognizing primers:    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 897 to nucleotide 916    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 1010 to nucleotide 1029    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 897 to nucleotide 914    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 899 to nucleotide 916    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 949 to nucleotide 966    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 1010 to nucleotide 1028    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 1008 to nucleotide 1027    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 1010 to nucleotide 1029    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 842 to nucleotide 861    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 842 to nucleotide 860    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 842 to nucleotide 862    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 842 to nucleotide 859    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 842 to nucleotide 863    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 920 to nucleotide 939    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 920 to nucleotide 937    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 1004 to nucleotide 1023    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 1004 to nucleotide 1024    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 1004 to nucleotide 1021    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 1004 to nucleotide 1025        c. 3′ flanking sequence recognizing primers:    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 432 to nucleotide 453    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 421 to nucleotide 442    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 433 to nucleotide 452    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 433 to nucleotide 453        d. foreign DNA sequence recognizing primers for use with 3′        flanking sequence recognizing primers:    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 1 to        nucleotide 20    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 18 to        nucleotide 37    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 20 to        nucleotide 39    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 14 to        nucleotide 33    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 17 to        nucleotide 37    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 19 to        nucleotide 37    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 21 to        nucleotide 40    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 21 to        nucleotide 39    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 50 to        nucleotide 67    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 71 to        nucleotide 90    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 74 to        nucleotide 93    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 96 to        nucleotide 115    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 13 to        nucleotide 33    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 15 to        nucleotide 33    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 16 to        nucleotide 37    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 19 to        nucleotide 39    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 20 to        nucleotide 37    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 20 to        nucleotide 40    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 22 to        nucleotide 39    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 22 to        nucleotide 40    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 28 to        nucleotide 47    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 42 to        nucleotide 61    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 70 to        nucleotide 90    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 72 to        nucleotide 92    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 72 to        nucleotide 91    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 73 to        nucleotide 93    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 75 to        nucleotide 93    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 75 to        nucleotide 94    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 340 to        nucleotide 359    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 424 to        nucleotide 443    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 16 to        nucleotide 33    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 18 to        nucleotide 39    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 19 to        nucleotide 40    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 27 to        nucleotide 47    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 29 to        nucleotide 47    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 37 to        nucleotide 56    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 40 to        nucleotide 59    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 41 to        nucleotide 59    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 42 to        nucleotide 59    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 69 to        nucleotide 90    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 71 to        nucleotide 91    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 72 to        nucleotide 93    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 73 to        nucleotide 91    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 73 to        nucleotide 90    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 74 to        nucleotide 94    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 94 to        nucleotide 115    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 189 to        nucleotide 208    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 339 to        nucleotide 359    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 341 to        nucleotide 359    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 358 to        nucleotide 377    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 366 to        nucleotide 385    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 424 to        nucleotide 445    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 26 to        nucleotide 47    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 30 to        nucleotide 47    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 36 to        nucleotide 56    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 38 to        nucleotide 56    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 39 to        nucleotide 59    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 41 to        nucleotide 60    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 70 to        nucleotide 91    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 73 to        nucleotide 94    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 74 to        nucleotide 91    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 338 to        nucleotide 359    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 342 to        nucleotide 359    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 357 to        nucleotide 377    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 359 to        nucleotide 377    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 367 to        nucleotide 385    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 367 to        nucleotide 386    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 35 to        nucleotide 56    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 38 to        nucleotide 59    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 356 to        nucleotide 377    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 360 to        nucleotide 377    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 364 to        nucleotide 385    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 366 to        nucleotide 386    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 368 to        nucleotide 385    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 368 to        nucleotide 386    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 407 to        nucleotide 428    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 40 to        nucleotide 61    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 365 to        nucleotide 384    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 369 to        nucleotide 386    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 422 to        nucleotide 443    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 367 to        nucleotide 387    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 366 to        nucleotide 387

As used herein, “the nucleotide sequence of SEQ ID No. Z from position Xto position Y” indicates the nucleotide sequence including bothnucleotide endpoints.

Preferably, the integration fragment has a length of between 50 and 500nucleotides, most preferably of between 100 and 350 nucleotides. Thespecific primers may have a sequence which is between 80 and 100%identical to a sequence within the 5′ or 3′ flanking region of the eliteevent and the foreign DNA of the elite event, respectively, provided themismatches still allow specific identification of the elite event withthese primers under optimized PCR conditions. The range of allowablemismatches however, can easily be determined experimentally and areknown to a person skilled in the art.

The following table exemplifies the sizes of expected DNA amplicons (orintegration fragments) with selected pairs of PCR primers.

Length Primer 1 From position Primer 2 To position amplicon GHI043 528SB327 829 302 GHI043 528 MAE080 1028 501 GHI043 528 MAE081 1214 687GHI043 528 GHI044 860 334 MAE078 1 MAE121 654 654 MAE070 96 MAE121 654559 MAE071 358 MAE121 654 297

Detection of integration fragments can occur in various ways e.g. viasize estimation after gel analysis. The integration fragments may alsobe directly sequenced. Other sequence specific methods for detection ofamplified DNA fragments are also known in the art.

As the sequence of the primers and their relative location in the genomeare unique for the elite event, amplification of the integrationfragment will occur only in biological samples comprising (the nucleicacid of) the elite event. Preferably when performing a PCR to identifythe presence of EE-GH3 in unknown samples, a control is included of aset of primers with which a fragment within a “housekeeping gene” of theplant species of the event can be amplified. Housekeeping genes aregenes that are expressed in most cell types and which are concerned withbasic metabolic activities common to all cells. Preferably, the fragmentamplified from the housekeeping gene is a fragment which is larger thanthe amplified integration fragment. Depending on the samples to beanalyzed, other controls can be included.

Standard PCR protocols are described in the art, such as in “PCRApplications Manual” (Roche Molecular Biochemicals, 2nd Edition, 1999).The optimal conditions for the PCR, including the sequence of thespecific primers, is specified in a “PCR identification protocol” foreach elite event. It is however understood that a number of parametersin the PCR identification protocol may need to be adjusted to specificlaboratory conditions, and may be modified slightly to obtain similarresults. For instance, use of a different method for preparation of DNAmay require adjustment of, for instance, the amount of primers,polymerase and annealing conditions used. Similarly, the selection ofother primers may dictate other optimal conditions for the PCRidentification protocol. These adjustments will however be apparent to aperson skilled in the art, and are furthermore detailed in current PCRapplication manuals such as the one cited above.

Alternatively, specific primers can be used to amplify an integrationfragment that can be used as a “specific probe” for identifying EE-GH3in biological samples. Contacting nucleic acid of a biological sample,with the probe, under conditions which allow hybridization of the probewith its corresponding fragment in the nucleic acid, results in theformation of a nucleic acid/probe hybrid. The formation of this hybridcan be detected (e.g. labeling of the nucleic acid or probe), wherebythe formation of this hybrid indicates the presence of EE-GH3. Suchidentification methods based on hybridization with a specific probe(either on a solid phase carrier or in solution) have been described inthe art. The specific probe is preferably a sequence which, underoptimized conditions, hybridizes specifically to a region within the 5′or 3′ flanking region of the elite event and preferably also comprisingpart of the foreign DNA contiguous therewith (hereinafter referred to as“specific region”). Preferably, the specific probe comprises a sequenceof between 50 and 500 bp, preferably of 100 to 350 bp which is at least80%, preferably between 80 and 85%, more preferably between 85 and 90%,especially preferably between 90 and 95%, most preferably between 95%and 100% identical (or complementary) to the nucleotide sequence of aspecific region. Preferably, the specific probe will comprise a sequenceof about 15 to about 100 contiguous nucleotides identical (orcomplementary) to a specific region of the elite event.

Oligonucleotides suitable as PCR primers for detection of the eliteevent EE-GH3 can also be used to develop a PCR-based protocol todetermine the zygosity status of the elite event. To this end, twoprimers recognizing the wild-type locus are designed in such a way thatthey are directed towards each other and have the insertion site locatedin between the primers. These primers may be primers specificallyrecognizing the 5′ and 3′ flanking sequences contained within SEQ ID NO1 or 2, respectively. This set of primers, together with a third primercomplementary to transforming DNA sequences and directed towards one ofthe flanking DNA, allow diagnostic PCR amplification of the EE-GH3specific locus, as well as of the wt locus. If the plant is homozygousfor the transgenic locus or the corresponding wt locus, the diagnosticPCR will give rise to a single PCR product typical, preferably typicalin length, for either the transgenic or wt locus. If the plant ishemizygous for the transgenic locus, two locus specific PCR productswill appear, reflecting both the amplification of the transgenic and wtlocus.

Furthermore, detection methods specific for elite event EE-GH3 whichdiffer from PCR based amplification methods can also be developed usingthe elite event specific sequence information provided herein. Suchalternative detection methods include linear signal amplificationdetection methods based on invasive cleavage of particular nucleic acidstructures, also known as Invader™ technology, (as described e.g. inU.S. Pat. Nos. 5,985,557 “Invasive Cleavage of Nucleic Acids”, 6,001,567“Detection of Nucleic Acid sequences by Invader Directed Cleavage,incorporated herein by reference). To this end, the target sequence mayhybridized with a labeled first nucleic acid oligonucleotide comprisingthe nucleotide sequence of SEQ ID No 1 from nucleotide 733 to nucleotideis 750 or its complement or said labeled nucleic acid probe comprisingthe nucleotide sequence of SEQ ID No 2 from nucleotide 413 to nucleotide430 or its complement and is further hybridized with a second nucleicacid oligonucleotide comprising the nucleotide sequence of SEQ ID No 1from nucleotide 715 to nucleotide 732 or its complement or said labelednucleic acid probe comprising the nucleotide sequence of SEQ ID No 2from nucleotide 431 to nucleotide 448 or its complement, wherein thefirst and second oligonucleotide overlap by at least one nucleotide. Theduplex or triplex structure which is produced by this hybridizationallows selective probe cleavage with an enzyme (Cleavase®) leaving thetarget sequence intact. The cleaved labeled probe is subsequentlydetected, potentially via an intermediate step resulting in furthersignal amplification.

A “kit” as used herein refers to a set of reagents for the purpose ofperforming the method of the invention, more particularly, theidentification of the elite event EE-GH3 in biological samples or thedetermination of the zygosity status of EE-GH3 containing plantmaterial. More particularly, a preferred embodiment of the kit of theinvention comprises at least one or two specific primers, as describedabove for identification of the elite event, or three specific primersfor the determination of the zygosity status. Optionally, the kit canfurther comprise any other reagent described herein in the PCRidentification protocol. Alternatively, according to another embodimentof this invention, the kit can comprise a specific probe, as describedabove, which specifically hybridizes with nucleic acid of biologicalsamples to identify the presence of EE-GH3 therein. Optionally, the kitcan further comprise any other reagent (such as but not limited tohybridizing buffer, label) for identification of EE-GH3 in biologicalsamples, using the specific probe.

The kit of the invention can be used, and its components can bespecifically adjusted, for purposes of quality control (e.g., purity ofseed lots), detection of the presence or absence of the elite event inplant material or material comprising or derived from plant material,such as but not limited to food or feed products.

As used herein, “sequence identity” with regard to nucleotide sequences(DNA or RNA), refers to the number of positions with identicalnucleotides divided by the number of nucleotides in the shorter of thetwo sequences. The alignment of the two nucleotide sequences isperformed by the Wilbur and Lipmann algorithm (Wilbur and Lipmann, 1983,Proc. Nat. Acad. Sci. USA 80:726) using a window-size of 20 nucleotides,a word length of 4 nucleotides, and a gap penalty of 4.Computer-assisted analysis and interpretation of sequence data,including sequence alignment as described above, can, e.g., beconveniently performed using the sequence analysis software package ofthe Genetics Computer Group (GCG, University of Wisconsin Biotechnologycenter). Sequences are indicated as “essentially similar” when suchsequences have a sequence identity of at least about 75%, particularlyat least about 80%, more particularly at least about 85%, quiteparticularly about 90%, especially about 95%, more especially about100%. It is clear than when RNA sequences are said to be essentiallysimilar or have a certain degree of sequence identity with DNAsequences, thymidine (T) in the DNA sequence is considered equal touracil (U) in the RNA sequence.

The term “primer” as used herein encompasses any nucleic acid that iscapable of priming the synthesis of a nascent nucleic acid in atemplate-dependent process, such as PCR. Typically, primers areoligonucleotides from 10 to 30 nucleotides, but longer sequences can beemployed. Primers may be provided in double-stranded form, though thesingle-stranded form is preferred. Probes can be used as primers, butare designed to bind to the target DNA or RNA and need not be used in anamplification process.

The term “recognizing” as used herein when referring to specificprimers, refers to the fact that the specific primers specificallyhybridize to a nucleic acid sequence in the elite event under theconditions set forth in the method (such as the conditions of the PCRidentification protocol), whereby the specificity is determined by thepresence of positive and negative controls.

The term “hybridizing” as used herein when referring to specific probes,refer to the fact that the probe binds to a specific region in thenucleic acid sequence of the elite event under standard stringencyconditions. Standard stringency conditions as used herein refers to theconditions for hybridization described herein or to the conventionalhybridizing conditions as described by Sambrook et al., 1989 (MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarbourLaboratory Press, NY) which for instance can comprise the followingsteps: 1) immobilizing plant genomic DNA fragments on a filter, 2)prehybridizing the filter for 1 to 2 hours at 42° C. in 50% formamide,5×SSPE, 2×Denhardt's reagent and 0.1% SDSi, or for 1 to 2 hours at 68°C. in 6×SSC, 2×Denhardt's reagent and 0.1% SDS, 3) adding thehybridization probe which has been labeled, 4) incubating for 16 to 24hours, 5) washing the filter for 20 min. at room temperature in 1×SSC,0.1% SDS, 6) washing the filter three times for 20 min. each at 68° C.in 0.2×SSC, 0.1% SDS, and 7) exposing the filter for 24 to 48 hours toX-ray film at −70° C. with an intensifying screen.

As used in herein, a biological samples is a sample of a plant, plantmaterial or products comprising plant material. The term “plant” isintended to encompass cotton (Gossypium hirsitum) plant tissues, at anystage of maturity, as well as any cells, tissues, or organs taken fromor derived from any such plant, including without limitation, any seeds,leaves, stems, flowers, roots, single cells, gametes, cell cultures,tissue cultures or protoplasts. “Plant material”, as used herein refersto material which is obtained or derived from a plant. Productscomprising plant material relate to food, feed or other products whichare produced using plant material or can be contaminated by plantmaterial. It is understood that, in the context of the presentinvention, such biological samples are tested for the presence ofnucleic acids specific for EE-GH3, implying the presence of nucleicacids in the samples. Thus the methods referred to herein foridentifying elite event EE-GH3 in biological samples, relate to theidentification in biological samples of nucleic acids which comprise theelite event.

As used herein “comprising” is to be interpreted as specifying thepresence of the stated features, integers, steps, reagents or componentsas referred to, but does not preclude the presence or addition of one ormore features, integers, steps or components, or groups thereof. Thus,e.g., a nucleic acid or protein comprising a sequence of nucleotides oramino acids, may comprise more nucleotides or amino acids than theactually cited ones, i.e., be embedded in a larger nucleic acid orprotein. A chimeric gene comprising a DNA sequence which is functionallyor structurally defined, may comprise additional DNA sequences, etc.

The present invention also relates to the development of an elite eventEE-GH3 in cotton to the plants comprising this event, the progenyobtained from these plants and to the plant cells, or plant materialderived from this event. Plants comprising elite event EE-GH3 wereobtained through as described in example 1.

Cotton plants or plant material comprising EE-GH3 can be identifiedaccording to the PCR identification protocol described for EE-GH3 inExample 2. Briefly, cotton genomic DNA present in the biological sampleis amplified by PCR using a primer which specifically recognizes asequence within the 5′ or 3′ flanking sequence of EE-GH3 such as theprimer with the sequence of SEQ ID NO: 3, and a primer which recognizesa sequence in the foreign DNA, such as the primer with the sequence ofSEQ ID NO: 11. DNA primers which amplify part of an endogenous cottonsequence are used as positive control for the PCR amplification. If uponPCR amplification, the material yields a fragment of the expected size,the material contains plant material from a cotton plant harboring eliteevent EE-GH3.

Plants harboring EE-GH3 are characterized by their glyphosate tolerance,which in the context of the present invention includes that plants aretolerant to the herbicide Glyphosate. Plants harboring EE-GH3 are alsocharacterized by having agronomical characteristics that are comparableto commercially available varieties of cotton in the US, in the absenceof weed pressure and use of glyphosate for weed control. It has beenobserved that the presence of a foreign DNA in the insertion region ofthe cotton plant genome described herein, confers particularlyinteresting phenotypic and molecular characteristics to the plantscomprising this event.

The current invention also provides genomic DNA comprising elite eventEE-GH3, as herein defined, which may be used as reference material inidentification kits.

The following examples describe the identification of elite event EE-GH3and the development of tools for the specific identification of eliteevent EE-GH3 in biological samples.

Unless otherwise stated, all recombinant DNA techniques are carried outaccording to standard protocols as described in Sambrook et al. (1989)Molecular Cloning. A Laboratory Manual, Second Edition, Cold SpringHarbour Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al.(1994) Current Protocols in Molecular Biology, Current Protocols, USA.Standard materials and methods for plant molecular work are described inPlant Molecular Biology Labfax (1993) by R. D. D. Croy published by BIOSScientific Publications Ltd (UK) and Blackwell Scientific Publications,UK.

In the description and examples, reference is made to the followingsequences:

-   SEQ ID No. 1: nucleotide sequence comprising a 5′ flanking region of    EE-GH3-   SEQ ID No. 2: nucleotide sequence comprising a 3′ flanking region of    EE-GH3-   SEQ ID No. 3: primer GH1043-   SEQ ID No. 4: primer SB327-   SEQ ID No. 5: primer MAE080-   SEQ ID No. 6: primer MAE081-   SEQ ID No. 7: primer MAE078-   SEQ ID No. 8: primer MAE070-   SEQ ID No. 9: primer MAE071-   SEQ ID No. 10: primer MAE121-   SEQ ID No. 11: primer GH1044-   SEQ ID No. 12: primer 1 for amplification of control fragment-   SEQ ID No. 13: primer 2 for amplification of control fragment

EXAMPLES 1. Identification of Elite Event EE-GH3

Herbicide-resistant cotton was developed by transformation of cottonwith a vector comprising the coding sequence of a modified epsps geneencoding a glyphosate tolerant EPSPS enzyme, under the control of apromoter of a histone gene as described in U.S. Pat. No. 5,491,288 orU.S. Pat. No. 5,792,930.

Elite event EE-GH3 was selected based on an extensive selectionprocedure based on good expression and stability of the herbicideresistance gene and its compatibility with optimal agronomiccharacteristics such as plant height, height to node, boll retention,stand, vigor, fiber length, fiber strength and lint yield wereevaluated.

The selected event was introduced into different commercial geneticbackgrounds, and results of field trials on different locations werecompared. Plants were sprayed with the herbicide using differenttreatments.

No visible damage as a result of herbicide application was ever observedafter application regardless of rate or stage of development at the timeof application. There were no detrimental effects on morphology orgrowth habit of plants by herbicide application

Furthermore, the event had normal leaf, flower and boll morphology,excellent fertility, and showed no disease or abnormal insectsusceptibility in multiple genetic backgrounds. During introgressioninto multiple genetic backgrounds no aberrant problems or abnormalitieswere observed.

2. Identification of the Flanking Regions of Elite Event EE-GH3

The sequence of the regions flanking the foreign DNA in the EE-GH3 eliteevent was determined using the thermal asymmetric interlaced (TAIL-) PCRmethod described by Liu et al. (1995, Plant J. 8(3):457-463). Thismethod utilizes three nested primers in successive reactions togetherwith a shorter arbitrary degenerate primer so that the relativeamplification efficiencies of specific and non-specific products can bethermally controlled. The specific primers were selected for annealingto the border of the foreign DNA and based on their annealingconditions. A small amount (5 μl) of unpurified, secondary and tertiary,PCR products were analyzed on a 1% agarose gel. The tertiary PCR productwas purified and sequenced.

2.1. Right (5′) Flanking Region

The fragment identified as comprising the 5′ flanking region obtained bythe TAIL-PCR method was sequenced (SEQ ID No. 1). The sequence betweennucleotide 1 and 732 corresponds to plant DNA, while the sequencebetween nucleotide 733 and 1214 corresponds to foreign DNA.

2.2. Left (3′) Flanking Region

The fragment identified as comprising the 3′ flanking region obtained bythe TAIL-PCR method was sequenced (SEQ ID No. 2). The sequence betweennucleotide 1 and 430 corresponds to foreign DNA, while the sequencebetween nucleotide 431 and 654 corresponds to plant DNA.

3. Development of a Polymerase Chain Reaction Identification Protocolfor EE-GH3 3.1. Primers

Specific primers were developed which recognize sequences within theelite event. More particularly, a primer was developed which recognizesa sequence within the 5′ flanking region of EE-GH3. A second primer wasthen selected within the sequence of the foreign DNA so that the primersspan a sequence of about 334 nucleotides. The following primers werefound to give particularly clear and reproducible results in a PCRreaction on EE-GH3 DNA:

GHI043: 5′-TTC.AgC.CgC.CAT.TgA.TgA.Ag-3′ (SEQ ID No.: 3) (target: plantDNA) GHI044: 5′-gTg.TAT.CCA.TgC.CTC.gAC.TC-3′ (SEQ ID No.: 11) (target:insert DNA)

Primers targeting an endogenous sequence are preferably included in thePCR cocktail. These primers serve as an internal control in unknownsamples and in the DNA positive control. A positive result with theendogenous primer-pair demonstrates that there is ample DNA of adequatequality in the genomic DNA preparation for a PCR product to begenerated. The endogenous primers were selected to recognize ahousekeeping gene in cotton:

GHI001: 5′-AAC.CTA.ggC.TgC.TgA.Agg.AgC-3′ (SEQ ID No.: 12) GHI002:5′-gTT.ACC.gTA.CAg.gTC.TTT.CC-3′ (SEQ ID No.: 13)

3.2. Amplified Fragments

The expected amplified fragments in the PCR reaction are:

For primer pair GHI001-GHI002: 445 bp (endogenous control)For primer pair GH1043-GHI044: 334 bp (EE-GH3 elite Event)

3.3. Template DNA

Template DNA was prepared from a leaf punch according to Edwards et al.(Nucleic Acid Research, 19, p 1349, 1991). When using DNA prepared withother methods, a test run utilizing different amounts of template shouldbe done. Usually 50 ng of genomic template DNA yields the best results.

3.4. Assigned Positive and Negative Controls

To avoid false positives or negatives, it was determined that thefollowing positive and negative controls should be included in a PCRrun:

-   -   Master Mix control (DNA negative control). This is a PCR in        which no DNA is added to the reaction. When the expected result,        no PCR products, is observed this indicates that the PCR        cocktail was not contaminated with target DNA.    -   A DNA positive control (genomic DNA sample known to contain the        transgenic sequences). Successful amplification of this positive        control demonstrates that the PCR was run under conditions which        allow for the amplification of target sequences.    -   A wild-type DNA control. This is a PCR in which the template DNA        provided is genomic DNA prepared from a non-transgenic plant.        When the expected result, no amplification of a transgene PCR        product but amplification of the endogenous PCR product, is        observed this indicates that there is no detectable transgene        background amplification in a genomic DNA sample.

3.5. PCR Conditions

Optimal results were obtained under the following conditions:

-   -   the PCR mix for 25 μl reactions contains:        -   2.5 μl template DNA        -   2.5 μl 10× Amplification Buffer (supplied by the            manufacturer with the Taq polymerase)        -   0.5 μl 10 mM dNTP's        -   0.5 μl GHI001 (10 pmoles/μl)        -   0.5 μl GHI002 (10 pmoles/μl)        -   0.25 μl GH1044 (10 pmoles/μl)        -   0.25 μl GH1043 (10 pmoles/μl)        -   0.1 μl Taq DNA polymerase (5 units/μl)        -   water up to 25 μl    -   the thermocycling profile to be followed for optimal results is        the following:

4 min. at 95° C. Followed by: 1 min. at 95° C. 1 min. at 57° C. 2 min.at 72° C. For 5 cycles Followed by: 30 sec. at 92° C. 30 sec. at 57° C.1 min. at 72° C. For 25 cycles Followed by: 10 minutes at 72° C.

3.6. Agarose Gel Analysis

To optimally visualise the results of the PCR it was determined thatbetween 10 and 20 μl of the PCR samples should be applied on a 1.5%agarose gel (Tris-borate buffer) with an appropriate molecular weightmarker (e.g. 100 bp ladder PHARMACIA).

3.7. Validation of the Results

It was determined that data from transgenic plant DNA samples within asingle PCR run and a single PCR cocktail should not be acceptableunless 1) the DNA positive control shows the expected PCR products(transgenic and endogenous fragments), 2) the DNA negative control isnegative for PCR amplification (no fragments) and 3) the wild-type DNAcontrol shows the expected result (endogenous fragment amplification).

When following the PCR Identification Protocol for EE-GH3 as describedabove, lanes showing visible amounts of the transgenic and endogenousPCR products of the expected sizes, indicate that the correspondingplant from which the genomic template DNA was prepared, has inheritedthe EE-GH3 elite event. Lanes not showing visible amounts of either ofthe transgenic PCR products and showing visible amounts of theendogenous PCR product, indicate that the corresponding plant from whichthe genomic template DNA was prepared, does not comprise the eliteevent. Lanes not showing visible amounts of the endogenous andtransgenic PCR products, indicate that the quality and/or quantity ofthe genomic DNA didn't allow for a PCR product to be generated. Theseplants cannot be scored. The genomic DNA preparation should be repeatedand a new PCR run, with the appropriate controls, has to be performed.

3.8. Use of Discriminating PCR Protocol to Identify EE-GH3

Before attempting to screen unknowns, a test run, with all appropriatecontrols, has to be performed. The developed protocol might requireoptimization for components that may differ between labs (template DNApreparation, Taq DNA polymerase, quality of the primers, dNTP's,thermocyler, etc.).

Amplification of the endogenous sequence plays a key role in theprotocol. One has to attain PCR and thermocycling conditions thatamplify equimolar quantities of both the endogenous and transgenicsequence in a known transgenic genomic DNA template. Whenever thetargeted endogenous fragment is not amplified or whenever the targetedsequences are not amplified with the same ethidium bromide stainingintensities, as judged by agarose gel electrophoresis, optimization ofthe PCR conditions may be required.

Leaf material from a number of cotton plants, some of which comprisingEE-GH3 were tested according to the above-described protocol. Samplesfrom elite event EE-GH3 and from cotton wild-type were taken as positiveand negative controls, respectively.

FIG. 2 illustrates the result obtained with the elite event PCRidentification protocol for EE-GH3 on a number of cotton plant samples.The samples in lanes 2 to 4 were found to contain the elite event as the334 bp band is detected, while the samples in lanes 5 to 12 do notcomprise EE-GH3. Lanes 5 to 11 comprise other cotton elite events(including plants comprising different glyphosate tolerance chimericgenes), lane 12 represents a non-transgenic cotton control; lane 13represents the negative control (water) sample, and lanes 1 and 14represent the Molecular Weight Marker (100 bp ladder).

4. Use of a Specific Integration Fragment as a Probe for Detection ofMaterial Comprising EE-GH3

A specific integration fragment of EE-GH3 is obtained by PCRamplification using specific primers GH1043 (SEQ ID No. 3) and GH1044(SEQ ID No. 11) or by chemical synthesis and is labeled. Thisintegration fragment is used as a specific probe for the detection ofEE-GH3 in biological samples. Nucleic acid is extracted from the samplesaccording to standard procedures. This nucleic acid is then contactedwith the specific probe under hybridization conditions which areoptimized to allow formation of a hybrid. The formation of the hybrid isthen detected to indicate the presence of EE-GH3 nucleic acid in thesample. Optionally, the nucleic acid in the samples is amplified usingthe specific primers prior to contact with the specific probe.Alternatively, the nucleic acid is labeled prior to contact with thespecific probe instead of the integration fragment. Optionally, thespecific probe is attached to a solid carrier (such as, but not limitedto a filter, strip or beads), prior to contact with the samples.

5. Protocol for the PCR-based Determination of the Zygosity Status ofEE-GH3 Cotton Plant Material 5.1. Primers

Two primers recognizing the nucleotide sequences of the wild-type locusprior to insertion of the elite event, were designed in such a way thatthey are directed towards each other and have the insertion site inbetween. This set of primers, together with a third primer complementaryto foreign DNA sequences and directed towards the flanking DNA, allowsimultaneous PCR amplification of the EE-GH3 locus as well as of the wtlocus.

The following primers were found to give particularly clear andreproducible results in a zygosity scoring PCR reaction on EE-GH3 DNA:

GHI043: 5′-TTC.AgC.CgC.CAT.TgA.TgA.Ag-3′ (SEQ ID No.: 3) (target: plantDNA of the 3′ flanking sequence) GHI044:5′-gTg.TAT.CCA.TgC.CTC.gAC.TC-3′ (SEQ ID No.: 11) (target: insert DNA)MAE121: 5′-CCA.TTC.CgA.TCA.TCg.AgT.Tg-3′ (SEQ ID No.: 10) (target: plantDNA of the 5′ flanking sequence)

5.2. Amplified Fragments

The expected amplified fragments in the PCR reaction are:

For primer pair GH1043-MAE121: 454 bp (corresponding to amplication ofwt locus)For primer pair GH1043-GHI044: 334 bp (EE-GH3 Elite Event)

5.3. Template DNA

Template DNA was prepared from a leaf punch according to Edwards et al.(Nucleic Acid Research, 19, p 1349, 1991). When using DNA prepared withother methods, a test run utilizing different amounts of template shouldbe done. Usually 50 ng of genomic template DNA yields the best results.

5.4. Assigned Positive and Negative Controls

To avoid false positives or negatives, it is advisable that thefollowing positive and negative controls should be included in a PCRrun:

-   -   Master Mix control (DNA negative control). This is a PCR in        which no DNA is added to the reaction. When the expected result,        no PCR products, is observed this indicates that the PCR        cocktail was not contaminated with target DNA.    -   A DNA positive control (genomic DNA sample known to contain the        transgenic sequences). Successful amplification of this positive        control demonstrates that the PCR was run under conditions which        allow for the amplification of target sequences.    -   A wild-type DNA control. This is a PCR in which the template DNA        provided is genomic DNA prepared from a non-transgenic plant.        When the expected result, no amplification of a transgene PCR        product but amplification of the endogenous PCR product, is        observed this indicates that there is no detectable transgene        background amplification in a genomic DNA sample.

5.5. PCR Conditions

Optimal results were obtained under the following conditions:

-   -   the PCR mix for 25 μl reactions contains:        -   x μl template DNA (150 ng)        -   2.5 μl 10× Amplification Buffer (supplied by the            manufacturer with the Taq polymerase)        -   0.5 μl 10 mM dNTP's        -   1.5 μl GH1043 (10 pmoles/μl)        -   1.0 μl GH1044 (10 pmoles/μl)        -   0.5 μl MAE121 (10 pmoles/μl)        -   0.1 μl Taq DNA polymerase (5 units/μl)        -   water up to 25 μl    -   the thermocycling profile to be followed for optimal results is        the following:

4 min. at 95° C. Followed by: 1 min. at 95° C. 1 min. at 57° C. 2 min.at 72° C. For 5 cycles Followed by: 30 sec. at 92° C. 30 sec. at 57° C.1 min. at 72° C. For 25 cycles Followed by: 10 minutes at 72° C.

5.6. Agarose Gel Analysis

To optimally visualise the results of the PCR it was determined thatbetween 10 and 20 μl of the PCR samples should be applied on a 1.5%agarose gel (Tris-borate buffer) with an appropriate molecular weightmarker (e.g. 100 bp ladder PHARMACIA).

5.7. Validation of the Results

Data from transgenic plant DNA samples within a single PCR run and asingle PCR Master Mix will not be acceptable unless:

-   -   the positive control shows the expected PCR products (transgenic        target amplification)    -   the wild-type-positive DNA control shows the expected result        (wild-type target amplification).    -   the negative control is negative for PCR amplification (no        fragments).

Lanes showing visible amounts of the transgenic PCR product of theexpected size and not showing visible amounts of the wild type PCRproduct, indicate that the corresponding plant from which the genomicDNA template was prepared, is homozygous for the transgenic genecassette.

Lanes showing visible amounts of the transgenic and wild type PCRproducts of the expected sizes, indicate that the corresponding plantfrom which the genomic template DNA was prepared, is hemizygous for thetransgenic gene cassette. Lanes not showing visible amounts of thetransgenic PCR product and showing visible amounts of the wild type PCRproduct, indicate that the corresponding plant from which the genomictemplate DNA was prepared, has not inherited the transgenic sequenceassayed for and is thus homozygous for the wild type locus.

Lanes not showing visible amounts of transgenic and wild type PCRproducts, indicate that the quality and/or quantity of the genomic DNAdidn't allow for a PCR product to be generated. These plants cannot bescored. The genomic DNA preparation should be repeated and a new PCRrun, with the appropriate controls, has to be performed.

5.8. Use of the Zygosity Scoring Protocol for Identification of ZygosityStatus in EE-GH3 Containing Plants.

FIG. 3 illustrates the result obtained with the zygosity scoring PCR forEE-GH3 on a number of cotton plant samples. The samples in lanes 2 to 8were found to contain the PCR fragment (334 bp) characteristic for eliteevent EE-GH3, while the samples in lanes 3, 5, 9 and 10 to 12 containedthe fragment characteristic for the presence of the wt locus. Lanes 2,4, 6, 7 and 8 therefore contain EE-GH3 in homozygous form, lanes 3 and 5contain EE-GH3 in hemizygous form and lane 9 contains the wt locus inhomozygous form (azygous for EE-GH3). Lane 10 represents anon-transgenic cotton control; lane 11 represents the negative control(water) sample, and lanes 1 and 12 represent the Molecular Weight Marker(100 bp ladder).

6. Introgression of EE-GH3 into Preferred Cultivars

Elite event EE-GH3 is introduced by repeated back-crossing intocommercial cotton cultivars such as but not limited to FM5013, FM5015,FM5017, FM989, FM832, FM966 and FM958, FM989, FM958, FM832, FM991,FM819, FM800, FM960, FM966, FM981, FM5035, FM5044, FM5045, FM5013,FM5015, FM5017 or FM5024.

It is observed that the introgression of the elite event into thesecultivars does not significantly influence any of the desirablephenotypic or agronomic characteristics of these cultivars (no linkagedrag) while expression of the transgene, as determined by glufosinatetolerance, meets commercially acceptable levels. This confirms thestatus of event EE-GH3 as an elite event.

As used in the claims below, unless otherwise clearly indicated, theterm “plant” is intended to encompass plant tissues, at any stage ofmaturity, as well as any cells, tissues, or organs taken from or derivedfrom any such plant, including without limitation, any seeds, leaves,stems, flowers, roots, single cells, gametes, cell cultures, tissuecultures or protoplasts.

Reference seed comprising elite event EE-GH3 was deposited as EE-GH3 atthe ATCC (10801 University Blvd., Manassas, Va. 20110-2209) on Jul. 19,2005, under ATCC accession number PTA-6878.

As used in the claims below, unless otherwise clearly indicated, theterm “plant” is intended to encompass plant tissues, at any stage ofmaturity, as well as any cells, tissues, or organs taken from or derivedfrom any such plant, including without limitation, any seeds, leaves,stems, flowers, roots, single cells, gametes, cell cultures, tissuecultures or protoplasts.

The above description of the invention is intended to be illustrativeand not limiting. Various changes or modifications in the embodimentsdescribed may occur to those skilled in the art. These can be madewithout departing from the spirit or scope of the invention.

1. A method for identifying elite event EE-GH3 in biological samplescomprising detecting an EE-GH3 specific region with a specific primer orprobe which specifically recognizes the 5′ or 3′ flanking region ofEE-GH3.
 2. The method of claim 1, wherein said method comprisesamplifying a DNA fragment of between 100 and 500 bp from a nucleic acidpresent in said biological samples using a polymerase chain reactionwith at least two primers, one of said primers recognizing the 5′flanking region of EE-GH3, said 5′ flanking region comprising thenucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 732or the 3′ flanking region of EE-GH3, said 3′ flanking region comprisingthe nucleotide sequence of the complement of SEQ ID No 2 from nucleotide431 to nucleotide 654, the other primer of said primers recognizing asequence within the foreign DNA comprising the nucleotide sequence ofthe complement of SEQ ID No. 1 from nucleotide 733 to nucleotide 1214 orthe nucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide430.
 3. The method of claim 2, wherein said primer recognizing the 5′flanking region consists of a nucleotide sequence of 17 to 200consecutive nucleotides selected from the nucleotide sequence of SEQ IDNo 1 from nucleotide 1 to nucleotide 732 or said primer recognizing the3′ flanking region of EE-GH3 consists of a nucleotide sequence of 17 to200 consecutive nucleotides selected from the nucleotide sequence of thecomplement of SEQ ID No 2 from nucleotide 431 to nucleotide 654, andsaid primer recognizing a sequence within the foreign DNA consists of 17to 200 consecutive nucleotides selected from the nucleotide sequence ofthe complement of SEQ ID No. 1 from nucleotide 733 to nucleotide 1214 orthe nucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide430.
 4. The method of claim 2, wherein said primer recognizing the 5′flanking region comprises at its extreme 3′ end a nucleotide sequence ofat least 17 consecutive nucleotides selected from the nucleotidesequence of SEQ ID No 1 from nucleotide 1 to nucleotide 732 or saidprimer recognizing the 3′ flanking region of EE-GH3 comprises at itsextreme 3′ end a nucleotide sequence of at least 17 consecutivenucleotides selected from the nucleotide sequence of the complement ofSEQ ID No 2 from nucleotide 431 to nucleotide 654, and said primerrecognizing a sequence within the foreign DNA comprises at its 3′ end atleast 17 consecutive nucleotides selected from the nucleotide sequenceof the complement of SEQ ID No. 1 from nucleotide 733 to nucleotide 1214or the nucleotide sequence of SEQ ID No 2 from nucleotide 1 tonucleotide
 430. 5. The method of claim 4, wherein said primerrecognizing the 5′ flanking region comprises of SEQ ID No. 3 and saidprimer recognizing a sequence within the foreign DNA comprises SEQ IDNo.
 11. 6. The method of claim 5, wherein said method comprisesamplifying a fragment of about 334 bp using the EE-GH3 identificationprotocol.
 7. A kit for identifying elite event EE-GH3 in biologicalsamples comprising one primer recognizing the 5′ flanking region ofEE-GH3, said 5′ flanking region comprising the nucleotide sequence ofSEQ ID No 1 from nucleotide 1 to nucleotide 732 or one primerrecognizing the 3′ flanking region of EE-GH3, said 3′ flanking regioncomprising the nucleotide sequence of the complement of SEQ ID No 2 fromnucleotide 431 to nucleotide 654, and one primer recognizing a sequencewithin the foreign DNA, said foreign DNA comprising the nucleotidesequence of the complement of SEQ ID No. 1 from nucleotide 733 tonucleotide 1214 or the nucleotide sequence of SEQ ID No 2 fromnucleotide 1 to nucleotide
 430. 8. The kit of claim 7, wherein saidprimer recognizing the 5′ flanking region consists of a nucleotidesequence of 17 to 200 consecutive nucleotides selected from thenucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 732or said primer recognizing the 3′ flanking region of EE-GH3 consists ofa nucleotide sequence of 17 to 200 consecutive nucleotides selected fromthe nucleotide sequence of the complement of SEQ ID No 2 from nucleotide431 to nucleotide 654, and said primer recognizing a sequence within theforeign DNA consists of 17 to 200 consecutive nucleotides selected fromthe nucleotide sequence of the complement of SEQ ID No. 1 fromnucleotide 733 to nucleotide 1214 or the nucleotide sequence of SEQ IDNo 2 from nucleotide 1 to nucleotide
 430. 9. The kit of claim 7, whereinsaid primer recognizing the 5′ flanking region comprises at its extreme3′ end a nucleotide sequence of at least 17 consecutive nucleotidesselected from the nucleotide sequence of SEQ ID No 1 from nucleotide 1to nucleotide 732 or said primer recognizing the 3′ flanking region ofEE-GH3 comprises at its extreme 3′ end a nucleotide sequence of at least17 consecutive nucleotides selected from the nucleotide sequence of thecomplement of SEQ ID No 2 from nucleotide 431 to nucleotide 654, andsaid primer recognizing a sequence within the foreign DNA comprises atits 3′ end at least 17 consecutive nucleotides selected from thenucleotide sequence of the complement of SEQ ID No. 1 from nucleotide733 to nucleotide 1214 or the nucleotide sequence of SEQ ID No 2 fromnucleotide 1 to nucleotide
 430. 10. The kit of claim 7, comprising aprimer consisting of the sequence of SEQ ID No. 3 and a primerconsisting of the sequence of SEQ ID No.
 11. 11. A primer comprising asequence which, under optimized detection conditions specificallyrecognizes a sequence within the 5′ or 3′ flanking region of EE-GH3,said 5′ flanking region comprising the nucleotide sequence of SEQ ID No1 from nucleotide 1 to nucleotide 732 and said 3′ flanking regioncomprising the nucleotide sequence of the complement of SEQ ID No 2 fromnucleotide 431 to nucleotide
 654. 12. The primer of claim 11, whereinsaid primer consists of a nucleotide sequence of 17 to 200 consecutivenucleotides selected from the nucleotide sequence of SEQ ID No 1 fromnucleotide 1 to nucleotide 732 or a nucleotide sequence of 17 to 200consecutive nucleotides selected from the nucleotide sequence of thecomplement of SEQ ID No 2 from nucleotide 431 to nucleotide
 654. 13. Theprimer of claim 11, wherein said primer comprises at its extreme 3′ enda nucleotide sequence of at least 17 consecutive nucleotides selectedfrom the nucleotide sequence of SEQ ID No 1 from nucleotide 1 tonucleotide 732 or a nucleotide sequence of at least 17 consecutivenucleotides selected from the nucleotide sequence of the complement ofSEQ ID No 2 from nucleotide 431 to nucleotide
 654. 14. A primercomprising at its extreme 3′ end the sequence of SEQ ID No.
 3. 15. Aprimer comprising at its extreme 3′ end the sequence of SEQ ID No. 11.16. The method of claim 1, wherein said method comprises hybridizing anucleic acid of biological samples with a specific probe for EE-GH3. 17.The method of claim 16, wherein the sequence of said specific probe hasat least 80% sequence identity with a sequence comprising part of the 5′flanking sequence or the 3′ flanking sequence of EE-GH3 and the sequenceof the foreign DNA contiguous therewith.
 18. The method of claim 17,wherein the sequence of said specific probe has at least 80% sequenceidentity with SEQ ID No. 1 from nucleotide 712 to 753 or SEQ ID No. 2from nucleotide 410 to 451, or the complement of said sequences.
 19. Akit for identifying elite event EE-GH3 in biological samples comprisinga specific probe, capable of hybridizing specifically to a specificregion of EE-GH3.
 20. The kit of claim 19, wherein the sequence of saidspecific probe has at least 80% sequence identity with a sequencecomprising part of the 5′ flanking sequence or the 3′ flanking sequenceof EE-GH3 and the sequence of the foreign DNA contiguous therewith. 21.The kit of claim 20, wherein the sequence of said specific probe has atleast 80% sequence identity with SEQ ID No. 1 SEQ ID No. 1 fromnucleotide 712 to 753 or SEQ ID No. 2 from nucleotide 410 to 451, or thecomplement of said sequences.
 22. A specific probe for theidentification of elite event EE-GH3 in biological samples.
 23. Theprobe of claim 22, wherein said probe has at least 80% sequence identitywith a sequence comprising part of the 5′ flanking sequence or the 3′flanking sequence of EE-GH3 and the sequence of the foreign DNAcontiguous therewith, or the complement thereof.
 24. The probe of claim23, wherein said probe has at least 80% sequence identity with SEQ IDNo. 1 from nucleotide 712 to 753 or SEQ ID No. 2 from nucleotide 410 to451, or the complement of said sequences.
 25. A specific probe for theidentification of elite event EE-GH3 in biological samples, the sequenceof being essentially similar to SEQ ID No. 1 from nucleotide 712 to 753or SEQ ID No. 2 from nucleotide 410 to 451, or the complement of saidsequences.
 26. A method for confirming seed purity comprising detectingan EE-GH3 specific region with a specific primer or probe whichspecifically recognizes the 5′ or 3′ flanking region of EE-GH3, in seedsamples.
 27. A method for screening seeds for the presence of EE-GH3comprising detecting an EE-GH3 specific region with a specific primer orprobe which specifically recognizes the 5′ or 3′ flanking region ofEE-GH3, in samples of seed lots.
 28. A method for determining thezygosity status of a plant, plant material or seed comprising eliteevent EE-GH3 comprising amplifying DNA fragments of between 100 and 500bp from a nucleic acid present in said biological samples using apolymerase chain reaction with at least three primers, one of saidprimers recognizing the 5′ flanking region of EE-GH3, said 5′ flankingregion comprising the nucleotide sequence of SEQ ID No 1 from nucleotide1 to nucleotide 732, a second of said primers specifically recognizingthe 3′ flanking region of EE-GH3, said 3′ flanking region comprising thenucleotide sequence of the complement of SEQ ID No 2 from nucleotide 431to nucleotide 654, a third of said primers recognizing a sequence withinthe foreign DNA comprising the nucleotide sequence of the complement ofSEQ ID No. 1 from nucleotide 733 to nucleotide 1214 or the nucleotidesequence of SEQ ID No 2 from nucleotide 1 to nucleotide
 430. 29. Themethod according to claim 28, wherein said first primers comprises thenucleotide sequence of SEQ ID No 3, said second primer comprises thesequence of SEQ ID No 10 and said third primer comprises the sequence ofSEQ ID No
 11. 30. A method of detecting the presence of elite eventEE-GH3 in biological samples through hybridization with a substantiallycomplementary labeled nucleic acid probe in which the probe:targetnucleic acid ratio is amplified through recycling of the target nucleicacid sequence comprising: a) hybridizing said target nucleic acidsequence to a first nucleic acid oligonucleotide comprising thenucleotide sequence of SEQ ID No 1 from nucleotide 733 to nucleotide 750or its complement or said first nucleic acid oligonucleotide comprisingthe nucleotide sequence of SEQ ID No 2 from nucleotide 413 to nucleotide430 or its complement; b) hybridizing said target nucleic acid sequenceto a second nucleic acid oligonucleotide comprising the nucleotidesequence of SEQ ID No 1 from nucleotide 715 to nucleotide 732 or itscomplement or said labeled nucleic acid probe comprising the nucleotidesequence of SEQ ID No 2 from nucleotide 431 to nucleotide 448 or itscomplement, wherein said first and second oligonucleotide overlap by atleast one nucleotide and wherein either said first or said secondoligonucleotide is labeled to be said labeled nucleic acid probe; c)cleaving only the labeled probe within the probe:target nucleic acidsequence duplex with an enzyme which causes selective probe cleavageresulting in duplex disassociation, leaving the target sequence intact;d) recycling of the target nucleic acid sequence by repeating steps (a)to (c); and e) detecting cleaved labeled probe, thereby determining thepresence of said target nucleic acid sequence.
 31. A transgenic cottonplant, or cells, parts, seed or progeny thereof, each comprising eliteevent EE-GH3 in its genome.
 32. (canceled)
 33. Seed comprising eliteevent EE-GH3 deposited at the ATCC under deposit number PTA-6878 orderivatives therefrom.
 34. A cotton plant or a part thereof, or seedtherefrom obtained from the seed of claim
 33. 35. (canceled)
 36. Acotton seed comprising elite event EE-GH3.
 37. A transgenic cottonplant, cell or tissue, comprising elite event EE-GH3, produced from theseed of claim
 36. 38. A method for producing a cotton plant or seedcomprising elite event EE-GH3 comprising crossing a plant of claim 31with another cotton plant, and planting the seed obtained from saidcross.
 39. Cotton genomic DNA comprising elite even EE-GH3.
 40. GenomicDNA produced from a cotton plant, plant cell or seed of claim 31.